Myotonic dystrophy (DM) is the most common adult-onset muscular dystrophy. Presently there is no effective treatment and, although the mutations are known, the pathomechanisms are incompletely understood. DM1 and DM2 are caused by different expansion mutations--(CTG) DM1 in DMPK and (CCTG) DM2 in ZNF9. Despite similar mutations, they are clinically distinct diseases. Since transcription of (CUG) DM1/(CCUG)DM2 is necessary and sufficient to cause disease, DM1 and DM2 are considered toxic RNA diseases. However, the only mechanism of toxicity extensively studied to date is aberrant splicing, and therapeutic efforts have focused mainly on this aspect. What is not known is the extent to which missplicing accounts for the symptoms and whether there are additional pathomechanisms. This knowledge gap limits understanding of the effectiveness of therapies that target missplicing. Our long-term goal is to understand the pathomechanisms underlying DM and to identify entry points for therapy. Evidence from animal models, particularly Znf9 mice, suggests that many DM2 features can be elicited by ZNF9 insufficiency, without (CCTG)/(CCUG)DM2 or aberrant splicing. We and others have demonstrated reduced ZNF9 mRNA and protein levels in DM2 patients. Therefore, ZNF9 loss is directly implicated in DM2 pathogenesis, but its role remains unknown. Our central hypothesis is that expression of (CCUG)DM2 in ZNF9 is toxic via multiple mechanisms. The objective of this application is to determine the role of ZNF9 in the pathology of DM2. The rationale for the proposed research is that since ZNF9 functions as a regulator of both transcription and translation, ZNF9 haploinsufficiency plays a critical role in DM2 by dysregulation of both transcriptional and translational targets. Because in mice ZNF9 haploinsufficiency alone results in a DM-like phenotype, we hypothesize that mutations in human ZNF9 can produce DM2-like symptoms. To test our hypotheses, we will pursue three specific aims: (1) Identify ZNF9 DNA binding sites and transcriptional targets; (2) Identify ZNF9 mRNA binding sites and translational targets; and (3) Sequence ZNF9 in patients with DM of unknown etiology, to identify mutations responsible for their DM-like phenotype. Aims 1 and 2 will utilize Next-Generation technology to sequence ZNF9- immunoprecipitated DNA and RNA complexes to identify ZNF9 targets. Candidate genes will be validated in muscle biopsies and cultures using RNA- and protein-based methods. In Aim 3 we will sequence ZNF9 in an already collected DM patient cohort without (CTG) DM1/(CCTG)DM2 expansions. The approach is innovative, because it utilizes global technologies and unique patient resources to investigate previously unrecognized pathomechanisms. The proposed research is significant because identification of a pathomechanism(s) independent of aberrant splicing will shift the existing paradigm, focused on splicing. Ultimately, such knowledge will provide new insights into the role of ZNF9 in DM2 and identify new candidate effector genes and cellular pathways as well as therapeutic targets, which will positively impact the quality of patient lives. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because it is ultimately expected to increase understanding of the role of ZNF9 and heretofore unrecognized mechanisms in the pathogenesis of myotonic dystrophy type 2 (DM2). Successful completion will result in the identification of new candidate effector genes and cellular pathways as well as targets for therapeutic intervention specific to myotonic dystrophy. Thus, the proposed research is relevant to the part of NIAMS/NINDS missions that pertains to developing fundamental knowledge that will help reduce the burdens of neuromuscular disease in human patients.